In the mammalian testis, interactions between the different somatic cell types (Sertoli, peritubular and Leydig) provide a unique microenvironment controlling spermatogenesis. In the past years, in vitro cultures of somatic testicular cells have become increasingly used in investigating the role of these cell types in supporting germ cell differentiation. However, these investigations are limited by the fact that none of the cell populations used are pure lines and the viability of these primary cultures seldom exceeds fifteen days, thus limiting the time of observation that is necessary for germ cell adaptation and differentiation.We have recently immortalized (using the SV40 large T antigen) all the cell types contributing to a developing seminiferous tubule in the mouse testis. Eight Sertoli, 16 peritubular and 22 Leydig cell lines have been established, cloned to purity by repeated limited dilutions, and cultured successfully for 90 generations in a period of 2.0 years. Under defined culture conditions 2 of the Sertoli lines and 3 out of 7 tested Leydig lines were found to express FSH and LH receptors respectively. More significantly, one germ cell line has been established that, based on its morphology, electronmicroscopic characteristics and its expression of the testicular isoform of cytochrome c and of the testis-specific lactate dehydrogenase (LDH-C) isozyme, has been characterized as corresponding to a transition between a spermatogonia B and a primary spermatocyte stage. Furthermore, these four immortalized cell types, when plated together, are able to reaggregate and reconstitute spermatogenic tubule-like structures in vitro. The availability of these cell lines represents a major breakthrough in our ability to study spermatogenesis in vitro, to identify testis-specific factors that control gene expression and to characterize the molecules that control gametogenesis. The overall aim of this project is to establish a co-culture system of functional Sertoli, Leydig and peritubular cell lines able to support germ cell differentiation in vitro. The specific aims of this proposal are: I. to characterize the functionality, hormonal responsiveness and steroidogenic potential, of the Sertoli, peritubular, and Leydig cell lines. II. to investigate the ability of the in vitro spermatogenic tubule to support normal germ cell differentiation. III. to investigate the ability of our immortalized germ cell line to differentiate in vitro. IV. to immortalize primordial germ cells and spermatogonia following the above or improved immortalization strategies and investigate their ability to differentiate in vitro.